Genome-wide transcriptional and functional analysis of human T lymphocytes treated with benzo[a]pyrene. Genome-wide transcriptional and functional analysis of human T lymphocytes treated with benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P), are widely distributed carcinogenic and immunotoxic environmental contaminants, known to affect T lymphocytes. Despite extensive studies conducted, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, B[a]P using a microarray-based transcriptome analysis. After a 48-h exposure to B[a]P, we identified 158 genes differentially expressed in T lymphocytes including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, and the proto-oncogene c-KIT but also other ones not previously shown to be targeted by B[a]P such as genes encoding the gap junction beta-2 (GJB2) and beta-6 (GJB6) proteins. Functional enrichment analysis revealed that those candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signalling pathways; it also revealed a clear alteration in essential biological functions, mainly those related to T lymphocyte recruitment. These data were confirmed by RT-qPCR assays for some selected genes such as the chemokine (C-C motif) ligand CCL3, and the cell adhesion protein like selectin L (SELL). Using functional tests in transwell migration experiments, B[a]P was shown to significantly decrease the chemokine (C-X-C motif) ligand 12 (CXCL12)-induced chemotaxis and transendothelial migration of T lymphocytes. Overall, the present study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, providing a broader understanding of the molecular basis of the immunotoxicity of PAHs. Overall design: Independent T lymphocyte cultures isolated from 16 blood donors, each available in CTR (dimethylsulfoxide) and B[a]P-treated conditions were performed. Equal amounts of RNAs were pooled from these cultures to constitute four equimolar pools with four different blood donors per pool. Each pool was hybridized to Human Clariom™ S GeneChip.

多环芳烃 (Polycyclic Aromatic Hydrocarbons, PAHs) 如苯并[a]芘 (Benzo[a]Pyrene, B[a]P)，是一类分布广泛的致癌且具有免疫毒性的环境污染物，已知可对T淋巴细胞 (T lymphocyte) 产生影响。尽管已有大量相关研究开展，但PAHs在人类T淋巴细胞中介导免疫毒性效应的分子靶点与信号通路仍未明确。本研究采用基于微阵列的转录组分析技术，对经典型PAH——苯并[a]芘处理的原代人类T淋巴细胞进行基因表达谱分析。在暴露于B[a]P 48小时后，我们共鉴定出158个在T淋巴细胞中差异表达的基因，其中既包括已知受PAHs调控的基因（如细胞色素P450 (Cytochromes P450, CYP) 1A1、1B1与原癌基因c-KIT (proto-oncogene c-KIT)），也包含此前未被证实可被B[a]P靶向的基因（如编码间隙连接蛋白β-2 (Gap Junction Beta-2, GJB2) 与β-6 (Gap Junction Beta-6, GJB6) 的基因）。功能富集分析显示，上述候选基因显著富集于芳香烃受体 (Aryl Hydrocarbon, AhR) 与干扰素 (Interferon, IFN) 信号通路；同时也揭示了T淋巴细胞核心生物学功能的显著紊乱，尤以T淋巴细胞募集相关功能为著。本研究通过实时定量聚合酶链反应 (Reverse Transcription Quantitative Polymerase Chain Reaction, RT-qPCR) 对部分筛选出的基因（如趋化因子 (C-C基序)配体CCL3 (Chemokine (C-C Motif) Ligand CCL3) 以及选择素L (Selectin L, SELL) 这类细胞黏附蛋白）进行了验证。通过穿室迁移实验的功能检测，我们证实B[a]P可显著降低趋化因子 (C-X-C基序)配体12 (Chemokine (C-X-C Motif) Ligand CXCL12) 诱导的T淋巴细胞趋化与跨内皮迁移能力。综上，本研究揭示了此前未被关注的PAHs响应通路——T淋巴细胞迁移，为阐明PAHs免疫毒性的分子机制提供了更全面的视角。实验设计概况：本研究从16名献血者体内分离得到独立的T淋巴细胞培养物，每份样本均设置二甲基亚砜 (Dimethylsulfoxide, DMSO) 对照与B[a]P处理组。将各培养物中的RNA等量混合，构建4个等摩尔混合样本库，每个样本库包含4名献血者的样本。每个样本库均与人类Clariom™ S基因芯片 (Human Clariom™ S GeneChip) 进行杂交。